The severity of the hearing impairment of otoferlin mutant mouse lines studied to date (Roux et al , 2006 ; Longo‐Guess et al , 2007 ; Pangrsic et al , 2010 ; Reisinger et al , 2011 ) has precluded further functional studies at the cellular, systems and behavioural level. To address this problem, we set out to generate a novel Otof mutant mouse model with an intermediate hearing defect, which would allow us to tackle current open questions regarding the function of otoferlin in synaptic sound encoding. We generated a knock‐in mouse carrying the p.Ile515Thr point mutation (in NP_001274418), which was identified in one OTOF allele in siblings suffering from severe to profound hearing loss when their body temperature rises to ≥ 38.1°C (Varga et al , 2006 ). At normal body temperature, patients had mild low‐frequency hearing loss, speech comprehension below the 10 th percentile both in quiet and noise and lacked ABRs. Later analysis revealed a premature STOP codon (Arg1116*) in the second OTOF allele (A. Starr, personal communication). The hearing disorder in Otof I515T / I515T mice largely recapitulates the phenotype described in human patients, except for the temperature sensitivity. Comprehensive analyses of synaptic sound encoding in Otof I515T / I515T mice from the molecular to the systems level indicate that otoferlin is critical for reformation of synaptic vesicles from endosome‐like intermediates and for the replenishment of the RRP. Finally, we provide a candidate molecular mechanism for temperature‐induced deafness in humans.

While most mutations in human OTOF lead to profound deafness, a defined set of missense mutations cause a striking non‐syndromic recessive temperature‐sensitive auditory synaptopathy (reviewed in Pangršič et al , 2012 ) that is exceptional in several aspects. First, at normal body temperature, patients have near‐normal pure tone hearing thresholds but impaired speech recognition, especially in background noise (Starr et al , 1998 ). Second, psychoacoustic testing of some patients revealed severe abnormalities of loudness adaptation to continuous pure tone stimulation, also called “auditory fatigue” (Wynne et al , 2013 ). Third, elevation of body temperature to ≥ 38.1°C due to physical activity or fever causes severe to profound deafness. In the five independent familial cases described so far, different missense mutations (Ile515Thr, Gly541Ser, Arg1607Trp and compound heterozygosity for Gly614Glu and Arg1080Pro) and an in‐frame deletion (Glu1804del) were discovered (Varga et al , 2006 ; Romanos et al , 2009 ; Marlin et al , 2010 ; Wang et al , 2010 ; Matsunaga et al , 2012 ). Furthermore, three more missense mutations in otoferlin (Pro1987Arg, Glu1700Gln and Ile1573Thr) were described to cause moderate age‐progressive hearing loss without evident temperature sensitivity (Varga et al , 2003 ; Chiu et al , 2010 ; Yildirim‐Baylan et al , 2014 ). Unfortunately, speech perception, auditory temporal processing, auditory fatigue and temperature sensitivity have not been tested on these patients so far.

Mutations in OTOF , the gene coding for the multi‐C 2 domain protein otoferlin, cause human prelingual deafness DFNB9 (Yasunaga et al , 1999 ). Otoferlin is required for a late step in exocytosis in mouse inner hair cells (IHCs), as its absence nearly abolishes depolarization‐induced exocytosis despite the presence of synaptic vesicles at the ribbon‐type active zones (Roux et al , 2006 ). It was proposed that this phenotype reflects a role of otoferlin as a Ca 2+ sensor of exocytosis (Roux et al , 2006 ; Johnson & Chapman, 2010 ), but this idea requires further experimental testing. Indeed, in the profoundly hearing impaired pachanga mouse model ( Otof Pga / Pga ), which carries a point mutation in otoferlin (Schwander et al , 2007 ), only sustained exocytosis is impaired, while fast exocytosis, reporting the fusion of the readily releasable pool (RRP), is not (Pangrsic et al , 2010 ). The finding of impaired vesicle replenishment led to the hypothesis that otoferlin also functions in vesicle priming, which was subsequently supported by a recent study showing a reduction in short tethers connecting synaptic vesicles with the active zone membrane in otoferlin knockout ( Otof −/− ) mice (Vogl et al , 2015 ). In addition to these putative roles in priming and fusion, otoferlin may be involved in exocytosis–endocytosis coupling via an interaction with the clathrin adapter protein AP‐2 (Duncker et al , 2013 ; Jung et al , 2015 ).

Results

The Ile515Thr mutation lowers otoferlin protein levels We introduced the Ile515Thr substitution in mouse Otof via targeted knock‐in (Appendix Supplementary Methods). First, we investigated the abundance and localization of otoferlin by using immunohistochemistry and confocal fluorescence microscopy in organs of Corti of 14‐ to 15‐day‐old (P14‐15) mice (Fig 1). Quantifying immunofluorescence per cell, we found a 65% reduction in otoferlin levels in OtofI515T/I515T IHCs compared to wild‐type (Otof+/+) controls (Fig 1A, B and G). In a parallel analysis of OtofPga/Pga IHCs, a 69% reduction of protein levels was detected (Fig 1C and G), close to previous results (73%, Pangrsic et al, 2010). The reduction of otoferlin in the mutant genotypes was confirmed using another anti‐otoferlin antibody (Appendix Fig S1A–D). The lower protein levels in OtofI515T/I515T IHCs are likely due to a faster degradation of mutated otoferlin (Appendix Fig S1E). In OtofI515T/I515T IHCs, the remaining otoferlin localized more towards the synaptic area below the midline of the nucleus of IHCs compared to Otof+/+, while it was found more apically in OtofPga/Pga IHCs (Fig 1H). In the IHCs of all genotypes, otoferlin immunoreactivity was found in the cytoplasm and at the plasma membrane. In OtofI515T/I515T IHCs, the otoferlin immunofluorescence at the plasma membrane relative to total cellular protein levels was unaltered as compared to Otof+/+ IHCs. In contrast, OtofPga/Pga IHCs showed an 85% lower relative membrane staining (Fig 1D–F and I). Taken together, the calculated absolute level of membrane‐bound otoferlin was reduced by 66% in OtofI515T/I515 and by 97% in OtofPga/Pga IHCs (Fig 1J). Figure 1.Otoferlin levels and cellular distribution are differentially affected in OtofI515T/I515T and OtofPgaPpga IHCs A–C. Immunofluorescence images (inverted intensity) of P14‐P15 IHCs from indicated genotypes, revealing differences in otoferlin fluorescence intensity and distribution. Maximum projection of confocal stacks; scale bar, 10 μm.

D–F. Upper panels, examples for IHCs, co‐labelled for otoferlin (magenta) and Vglut3 (green) and position of the line for line scans; maximum projection of few optical sections; scale bars, 5 μm. Lower panels, for quantification of membrane staining, the fluorescence was normalized to the cellular fluorescence for each fluorophore, and then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values (black line) was used to determine the position of the basal membrane. At the most basal cellular point along this line which exceeds the threshold value of 2 (yellow diamond), the otoferlin‐Vglut3 fluorescence difference (blue line) gave the value for relative otoferlin plasma membrane levels (orange diamond). Insets: enlargements of basal regions.

G. Otoferlin protein levels were reduced in Otof I515T / I515T IHCs (indicated numbers represent numbers of cells) and Otof Pga / Pga IHCs compared to wild‐type ( Otof +/+ ) controls (mean ± SEM).

H. Ratio of apical/basal protein levels (above/below nuclear midline depicted as green line in (A) indicates an apical shift of otoferlin in Otof Pga / Pga IHCs.

I. Relative levels of membrane‐bound otoferlin at the basal pole of IHCs (mean ± SEM).

J. Absolute amount of otoferlin at the basal IHC plasma membrane, gained by multiplication of relative plasma membrane levels (I) × total cellular otoferlin protein levels (G) (mean ± SEM). Data information: Cell numbers in (G) apply also to (H–J); Kruskal–Wallis test; ***P < 0.001. Data information: Cell numbers in (G) apply also to (H–J); Kruskal–Wallis test; ***< 0.001. We conclude that the Ile515Thr mutation lowers the absolute otoferlin levels, but preserves the relative distribution between plasma membrane and cytoplasm.

Auditory brainstem responses (ABRs) indicate a progressive hearing impairment in OtofI515T/I515T mice Hearing was first assessed by ABR recordings. ABR thresholds were elevated by 20 dB for pure tones and 10 dB for click stimuli in young OtofI515T/I515T mice (Fig 2A). The amplitude of the spiral ganglion neuron (SGN) compound action potential, approximated as the amplitude of ABR wave I in response to clicks, was reduced to one‐third of the Otof+/+ littermate values, while subsequent waves were better preserved (Figs 2B and C, and EV1A and B). Together, population responses indicate a mild impairment of synchronous auditory signalling already in juvenile mice. We then cross‐bred OtofI515T/I515T mice with deaf Otof−/− mice in order to model the human OTOFI515T/R1116* subjects (Varga et al, 2006) even more closely. We found an additional elevation of ABR thresholds by 15 dB and a further reduction of ABR amplitudes in Otof−/I515T mice, indicating a gene dosage‐dependent effect (Figs 2A–C and EV1A). Consistent with a primary defect of the IHC synapse, distortion product otoacoustic emissions (DPOAE) were present, which report active cochlear amplification and require intact mechanoelectrical transduction (Fig EV1C). Figure 2.Hearing, assessed by ABR, is impaired due to the Ile515Thr mutation in otoferlin ABR thresholds in OtofI515T/I515T (red, n = 5) and Otof−/I515T (blue, n = 7) mice were elevated compared to Otof+/+ mice (black, n = 5) at an age of 3–4 weeks. The grey dotted line indicates the maximum loudspeaker output of 90 dB; thresholds exceeding this value were set to 100 dB for calculation of the mean ± SEM. At 12 kHz, only the threshold increase for Otof−/I515T versus Otof+/+ is significant (Kruskal–Wallis test with Dunn's multiple comparisons test between all three groups). Grand averages of ABR waveforms ± SEM in response to 80 dB click stimulation of the mice analysed in (A): The small wave preceding ABR wave I probably represents the summating potential (SP, hair cell receptor potential), which is intact in Otof mutants. ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T/I515T mice (Fig EV1). Mean ABR wave I amplitude ± SEM for different stimulus intensities (all differences between genotypes are significant; two‐way ANOVA with Tukey's multiple comparison test). At 8 weeks (circles) and 25 weeks (open squares), OtofI515T/I515T mice showed highly elevated ABR thresholds compared to Otof+/+ mice (n = 7–8 each; P < 0.001 at 12 kHz, Mann–Whitney U‐test). Grey dotted line as in (A). Grand averages of ABR waveforms ± SEM in response to 80 dB click stimulation in mice aged 8 weeks. OtofI515T/I515T (n = 8) have drastically reduced ABR amplitudes compared to Otof+/+ mice (n = 7). Otof−/− mice have no ABR (green, n = 9). Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8‐week‐old and 25‐week‐old OtofI515T/I515T and Otof+/+ mice (P < 0.001, two‐way ANOVA). Click here to expand this figure. Figure EV1.Partial central compensation of ABR wave I amplitude reduction, otoferlin protein levels during ageing and intact cochlear amplification Amplitude growth of ABR waves II‐IV with rising stimulus intensity to click stimulation in OtofI515T/I515T (red, n = 5), Otof−/I515T (blue, n = 6) and Otof+/+ (black, n = 5) animals tested at an age of 3–4 weeks, indicating a severe deficit of synchronous spiral ganglion neuron activation in Otof mutants but partial compensation in the auditory brainstem (mean ± SEM). Latencies of ABR waves I–IV from the same data set. Left: Interpolated DPOAE thresholds determined as the F1 intensity at which DPOAE intensity reached −10 dB SPL are comparable between OtofI515T/I515T (red) and Otof+/+ (black) mice. Right: Amplitude growth of DPOAE in response to pairs of sine waves (frequencies indicated above each panel) at rising intensities. F1 intensity was always 10 dB above F2 intensity. Otoferlin protein levels were even further reduced during ageing in OtofI515T/I515T IHCs, explaining the age‐dependent decrease in ABR amplitudes. Numbers indicate number of IHCs analysed (Tukey–Newman–Keuls test for parametric multiple comparisons; ***P < 0.001). Representative images used for synapse counting in OtofI515T/I515T (top) and Otof+/+ (bottom) IHCs. For 2‐ to 3‐week‐old mice (left), z‐projections of confocal stacks stained for ribbons (CtBP2, red) and postsynaptic glutamate receptors (GluR2/3, green) are shown. For 9‐ to 16‐week‐old mice (right), single sections of IHCs imaged in z‐stacks, stained for ribbons (CtBP2, red), the cytoplasmatic calcium buffer parvalbumin (green) and SGNs stained for Na/K‐ATPase (blue) are shown. Quantification of synapses; numbers indicate the number of IHCs analysed; mean ± SEM. Like in some patients with age‐progressive hearing loss due to OTOF mutations (Varga et al, 2003; Chiu et al, 2010; Yildirim‐Baylan et al, 2014), the hearing impairment, reflected by altered ABR thresholds and amplitudes, progressed rapidly during adolescence (Fig 2D–F), which correlated with a further reduction in otoferlin protein levels (Fig EV1D).

Intact synaptic vesicle fusion but impaired vesicle replenishment in OtofI515T/I515T IHCs Perturbations of otoferlin function have been shown to interfere with presynaptic function in IHCs (Roux et al, 2006; Pangrsic et al, 2010). To test the effect of the Ile515Thr mutation, we performed perforated‐patch recordings from IHCs in P14‐P17 mice at room temperature (RT). The current–voltage relationship revealed a normal voltage dependence of activation and amplitude of Ca2+ currents in OtofI515T/I515T IHCs (Fig 3A and B). We then measured exocytosis as increments of plasma membrane capacitance (ΔC m ) in response to step depolarizations to the voltage where maximal Ca2+ currents are elicited (typically −14 mV). Depolarizations were followed by recovery periods of 30 to 60 s at −84 mV, which precludes exocytosis triggered by voltage‐gated Ca2+ influx. Comparable ΔC m in response to short depolarizations (2–20 ms) indicated intact fusion of a normally sized RRP under these experimental conditions (Fig 3C and D). Consistently, the number of ribbon synapses was normal in mutant IHCs (Fig EV1E and F). However, when OtofI515T/I515T IHCs were depolarized for 50 ms or longer, exocytosis was significantly smaller than in controls (Fig 3C and D). Such sustained exocytosis is thought to reflect replenishment of synaptic vesicles to the RRP and their subsequent fusion, and/or active zone clearance (Pangršič et al, 2012). The rate of sustained exocytosis was reduced to ~340 vesicles/s/active zone, compared to ~700 vesicles/s/active zone in Otof+/+ IHCs (see Appendix Supplementary Methods), but was still considerably faster than in OtofPga/Pga IHCs (~200 vesicles/s/active zone, Pangrsic et al, 2010). Figure 3.Sustained exocytosis is impaired in OtofI515T/I515T hair cells A, B. No difference in voltage‐dependent Ca 2+ currents (A) and fractional activation of I Ca channels (B) between Otof I515T / I515T IHCs ( n = 16) and IHCs of Otof +/+ littermates ( n = 13; mean ± SEM).

C. Exocytosis was recorded by measures of changes in membrane capacitance (ΔCm, lower panel) in response to depolarization (left, 20 ms; right, 100 ms) to the voltage where maximum Ca 2+ currents were elicited (upper panel), typically −14 mV. Representative examples.

D. Upper panel, while for stimuli up to 20 ms exocytosis from Otof I515T / I515T ( n = 13) and Otof +/+ ( n = 11) IHCs was similar, sustained exocytosis, representing most likely the release of replenished vesicles, was significantly reduced in Otof I515T / I515T IHCs (mean ± SEM; t ‐test; ** P < 0.01; *** P < 0.001). Lower panel, Ca 2+ current integrals were of similar size (mean ± SEM).

E. Flash photolysis of caged Ca 2+ elicited a smaller exocytic response in Otof I515T / I515T IHCs (mean ± SEM).

F. OtofI515T/I515T IHCs and Otof+/+ littermates (black circles). Open circles represent previously published data on IHCs of hearing wild‐type mice (Beutner et al, 2001 et al, 2010 The kinetics of the fast component from (E) was comparable betweenIHCs andlittermates (black circles). Open circles represent previously published data on IHCs of hearing wild‐type mice (Beutner; Pangrsic). In order to test the fusion kinetics of RRP vesicles in OtofI515T/I515T IHCs, we recorded ΔC m in response to fast Ca2+ uncaging by UV laser. Here, exocytosis was comparable in kinetics between OtofI515T/I515T and Otof+/+. The amplitude was significantly reduced in OtofI515T/I515T IHCs (Fig 3E and F; fast and slow components reduced to 40 and 63%, respectively). This indicates that the Ile515Thr mutation does not impair the Ca2+‐triggered fusion of vesicles to the plasma membrane itself, but instead impairs vesicle replenishment, potentially affecting priming, active zone clearance or yet another mechanism.

In vivo postsynaptic recordings reveal a use‐dependent depression of sound encoding at OtofI515T/I515T synapses In order to directly assess sound encoding at OtofI515T/I515T synapses in vivo, we performed extracellular recordings from individual SGNs, each driven by a single IHC active zone (Fig 4). We found spontaneous spiking (Fig 4A), sound thresholds and frequency tuning (Fig EV2A and B) of individual SGNs to be normal which corroborates our notion of intact cochlear amplification in OtofI515T/I515T mice. Upon stimulation with tone bursts at a stimulus rate of 2 Hz, OtofI515T/I515T SGNs showed a near‐normal onset response with a high rate of instantaneous spiking, but then underwent stronger spike rate adaptation, not reaching a steady state within the 50 ms of stimulation (Fig 4B and E; ratio of onset/adapted rates 5.2 ± 1.8 in OtofI515T/I515T SGNs versus 3.5 ± 0.2 in Otof+/+ SGNs, P = 0.03, t‐test). Compared to SGNs from Otof+/+ littermates, the time course of adaptation was slower (Tau 10.9 ± 3.0 ms versus 6.1 ± 1.7 ms, single exponential fit, P < 0.001, t‐test). Figure 4.Enhanced adaptation and slowed recovery of SGN spiking in OtofI515T/I515T A. Spontaneous rates of SGNs from Otof I515T / I515T (red, n = 35) and Otof +/+ littermates (black, n = 39) were not significantly different ( P = 0.83, Kolmogorov–Smirnov test).

B–D. Averaged poststimulus time histograms ± SEM from Otof I515T / I515T mice ( n = 25–32) and Otof +/+ littermate SGNs ( n = 13–27) to stimulation with 50 ms tone bursts at the characteristic frequency (CF) of each fibre, 30 dB above threshold at indicated stimulus rates.

E. Quantification of onset (largest 0.5 ms bin, Mann–Whitney U ‐test) and adapted responses (averaged 35–45 ms from response onset, *** P < 0.001, t ‐test, Tukey quartile box plot) from data in (B–D).

F. The jitter of the first sound‐evoked spike was significantly increased ( P < 0.001, Mann–Whitney U ‐test) but retained its inverse correlation with spike rates.

G. Spike rate increases with rising stimulus intensity were significantly shallower in Otof I515T / I515T SGNs, both for repetitive stimulation with 50 ms tone bursts (left, P = 0.014, t ‐test) and for continuous stimulation with amplitude‐modulated tones (right, P < 0.001, Mann–Whitney U ‐test). Lines represent mean ± SEM.

H. Phase locking to amplitude‐modulated tones (assessed as the maximal synchronization index) was typically less precise in Otof I515T / I515T SGNs than in Otof +/+ SGNs ( P = 0.09, Mann–Whitney U ‐test).

I. 100 ms masker tone and 15 ms probe tones (both at CF, 30 dB above threshold) were separated by silent intervals of variable duration. Inter‐masker intervals were 500 ms for Otof +/+ and 1,000 ms for Otof I515T / I515T . The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in Otof I515T / I515T (pink; mean ± SEM red) compared to Otof +/+ (grey; mean ± SEM black; for 4 ms interval: P = 0.001, t ‐test) and a slowed time course of recovery (x: half‐time of recovery, interpolated from normalized recovery functions; P < 0.001, Mann–Whitney U ‐test).

J. OtofI515T/I515T mice (pink, mean ± SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ± SEM black) for shorter gap durations. See also Fig Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5mice (pink, mean ± SEM red) avoided drinking less efficiently than 2mice (grey, mean ± SEM black) for shorter gap durations. See also Fig EV3 Click here to expand this figure. Figure EV2.Single SGN responses show normal frequency tuning and thresholds, but abnormalities of latencies and spike rates Representative examples of SGN tuning curves from OtofI515T/I515T (pink/red) and Otof+/+ (grey/black) mice. Thresholds at CF were comparable in OtofI515T/I515T (red) and Otof+/+ (black) SGNs. The median first spike latency (FSL) in response to 200 repetitions of tone bursts at CF, 30 dB above threshold at a stimulus rate of 5 Hz was significantly prolonged in OtofI515T/I515T (red closed circles) and Otof+/+ (black closed circles) (P = 0.0003, t‐test). However, when the expected median FSL for the respective onset spike rate was subtracted (open symbols), FSLs were normal. The expected median FSL was calculated by the following formula which was derived from a large population of wild‐type SGNs: −2.5 × log(onset rate) + 20.23. All FSL estimates were first corrected for the system delay (3.6 ms), for the time of the 4 ms tone burst ramp to reach fibre threshold and for the travelling wave delay (up to 0.8 ms according to our wild‐type data set). Spike rate increases in individual OtofI515T/I515T (red) SGNs in response to tone burst stimulation at CF and varying intensities were shallower than in Otof+/+ (black) SGNs. The sound intensity refers to the threshold of each SGN, defined by a spike rate increase of 20 Hz over spontaneous rate. Their dynamic range (the range of intensities over which the spike rate increased from 10% to 90% of the evoked rate) was normal. Spike rate increases in individual OtofI515T/I515T (red) SGNs in response to continuous presentation of amplitude‐modulated sounds (CF, varying intensities, referenced to threshold like in D, modulation frequency 500 Hz) were much shallower than in Otof+/+ (black) SGNs. At higher stimulus rates of 5 or 10 Hz, both onset and adapted spike rates decreased dramatically (Fig 4C–E). Consistent with the reduced spiking at sound onset, the first spikes then also showed a highly significant increase in latency (Fig EV2C) and jitter (Fig 4F). As evoked spike rates were low and the dynamic range unchanged, rate‐intensity functions of individual SGNs were shallower than normal (Figs 4G and EV2D and E). The spike rates and precision of spike timing were even further decreased when continuous amplitude‐modulated sound stimuli were applied (Figs 4G and H, and EV2F). The enhanced adaptation and slowed recovery from adaptation were also obvious from responses to paired stimuli (Fig 4I) where the half‐time of recovery from forward masking was fivefold increased from 28.7 ± 5.6 ms in 9 Otof+/+ to 157.5 ± 40.5 ms in 13 OtofI515T/I515T SGNs (P < 0.001, Mann–Whitney U‐test). In summary, OtofI515T/I515T SGNs show an unusual sound encoding deficit that is dominated by a use‐dependent reduction of sound‐evoked spiking, likely resulting from impaired replenishment of the RRP and/or impaired active zone clearance.

Impaired synaptic sound encoding in OtofI515T/I515T mice affects the perception of silent gaps in noise Using prepulse inhibition of the acoustic startle response, we found an impairment of gap detection performance in OtofI515T/I515T mutants compared to Otof+/+ littermates (Fig EV3A–C). We propose that this is consistent with the delayed recovery from adaptation (Fig 4I) and reflects the impaired vesicle replenishment during the gap. For a more sensitive method that approaches the physiological limit of gap detection abilities, we then employed operant conditioning using the Audiobox system (de Hoz & Nelken, 2014). We conditioned mice to attempt to drink water only when continuous broadband noise was present. When the noise was interrupted by 90 ms silent gaps, access to the water bottles was denied and drink attempts were punished by air puffs. After reaching > 30% discrimination performance, we introduced shorter gaps in a total of 8% of the trials. The two Otof+/+ mice avoided drinking when gaps lasted 3 ms or more. This agrees well with descriptions of gap thresholds near 2 ms in CBA/J mice (Radziwon et al, 2009). In contrast, OtofI515T/I515T often attempted to drink in trials with short gaps (Figs 4J and EV3F). The interpolated 50% value of the normalized discrimination function was 2.7 ± 0.4 ms in Otof+/+ mice and 17.2 ± 4.9 ms in OtofI515T/I515T. Click here to expand this figure. Figure EV3.Hearing impairment was assessed by startle responses and operant conditioning of mice A, B. Acoustic startle responses to stimulation with 10 ms noise (A) or 12 kHz tone (B) bursts in Otof I515T / I515T (10 individuals pink, mean ± SEM red) and Otof +/+ (9 individuals grey, mean ± SEM black) animals backcrossed to a CBA/J strain background.

C. Prepulse inhibition of the startle response induced by silent gaps of varying duration was stronger in wild‐type (eight individuals grey, mean ± SEM black) than in Otof I515T / I515T mice (eight individuals pink, mean ± SEM red). Gap detection thresholds were determined as the minimal duration of silent gaps in 70 dB background noise that were effective in significantly attenuating the amplitude of startle response elicited by 115 dB 12 kHz tone bursts (Mann–Whitney U ‐test followed by Bonferroni correction). Gap thresholds (squares) were elevated in Otof I515T / I515T mice ( P = 0.04, Mann–Whitney U ‐test). In the control condition (no gap), the mean median startle amplitude for Otof I515T / I515T (0.07 ± 0.02) was identical to Otof +/+ (0.07 ± 0.02; P = 0.99, t ‐test).

D. Two Otof I515T / I515T mice (red) and one Otof +/+ mouse (black) learned to avoid drinking when they heard 12 kHz tone burst stimuli (400 ms, 3 Hz stimulus rate, 80 dB) during their visits to the soundproof corner of the “IntelliCage” operant conditioning system and drank water only when they did not perceive sounds. All three mice reacted to stimuli at 20 and 30 dB like for silence and for stimuli above 30 dB like for 80 dB, indicating comparable hearing thresholds near 35 dB for all three mice at the age of 14–21 weeks.

E. Subjective hearing thresholds in the same mice from (D) increased by approximately 5–15 dB when the mice were retested at an age of 42–53 weeks.

F. Behavioural gap detection did not show clear age‐related changes in a wild‐type mouse tested at 15–23 and 40–55 weeks. In a second task, we conditioned mice to avoid drinking when they heard 12 kHz tone bursts. The responses to varying tone intensities indicate comparable hearing thresholds in two OtofI515T/I515T mice and one Otof+/+ mouse (Fig EV3D and E). Together, the impaired gap detection performance and normal sound sensitivity in OtofI515T/I515T mice are consistent with the results of in vivo recordings from SGNs that show normal sound sensitivity but impaired recovery from adaptation. Like in other mice with synaptic transmission deficits (e.g. Buran et al, 2010; Jung et al, 2015) and patients with auditory synaptopathy/neuropathy, the deficit appears overly pronounced in ABR recordings which represent a combination of the sensitivity, the rate and the synchronicity of SGN firing.

ABR amplitudes gradually decline at elevated temperature Our data show that OtofI515T/I515T mice reflect the auditory phenotype of human patients at normal body temperature very well. Thus, we assume that the synaptic disease mechanisms found in mice at or below physiological temperature likely describe the situation in human patients. We next assayed whether mice, like the human patients, experience an exacerbation of their hearing loss when their body temperature exceeds 38°C (Varga et al, 2006). We monitored the amplitude of ABR wave I in response to click stimulation while locally applying heat to the inner ear (Fig 5A–C and Appendix Fig S2). Here, we found a reversible linear decrease in ABR wave I amplitude in both OtofI515T/I515T and Otof+/+ littermate mice, with comparable average regression slopes of 0.084 μV/°C and 0.081 μV/°C, respectively. The effect varied considerably between animals, presumably due to variable experimental conditions. In some animals (3/6 OtofI515T/I515T and 2/6 Otof+/+), ABR amplitudes did not recover after extensive heating (Appendix Fig S2B and C), presumably indicating permanent heat damage. Nonetheless, since ABRs in the mouse model never disappeared completely, we conclude that the elevated cochlear temperature did not fully abolish sound encoding in OtofI515T/I515T mouse mutants, as it seems to be the case in human OtofI515T/R1116* patients. Thus, as the heat‐induced phenotype seems to be weaker in mice compared to human patients, the cell physiological or ultrastructural effect of fever found in our mouse model might also be weaker than in human patients. Figure 5.Apparently lower thermal sensitivity of hearing in mice than in humans, likely due to a human RXR motif reducing plasma membrane localization of Ile515Thr‐otoferlin A. Exemplary ABR traces (click 100 dB) from one Otof I515T / I515T mouse recorded at indicated bulla temperatures during local heating. Note that ABRs never disappeared completely, but wave I amplitude changed reversibly with temperature.

B, C. ABR wave I amplitudes in Otof I515T / I515T (B, click 100 dB) and Otof +/+ mice (C, click 80 dB) decreased with increasing temperature in the bulla. Each colour represents a different experiment, and dashed lines are line fits. Open symbols indicate the beginning of the experiments; subsequent recordings are connected by lines. The four indicated data points in (B) correspond to ABRs in (A).

D. Explanted organ of Corti from an Otof +/+ mouse at P4 after 2 days in vitro (DIV2) at 37°C immunostained against otoferlin (magenta) and Vglut3 (green). Note the intense immunostaining of otoferlin at the plasma membrane. Scale bar, 10 μm.

E–I. Otof −/− mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2. (E) Example cell transfected with wild‐type mouse otoferlin cDNA. (F, G) Representative cells transfected with mouse otoferlin including the 20 amino acids of the RXR motif (see Fig Explanted organs of Corti frommice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2. (E) Example cell transfected with wild‐type mouse otoferlin cDNA. (F, G) Representative cells transfected with mouse otoferlin including the 20 amino acids of the RXR motif (see Fig EV4 ). Cells in (G) were incubated at 38.5°C for 30 min prior to fixation. The RXR‐otoferlin transfected cells (F, G) show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence/eGFP fluorescence at the basal pole of the cells (arrows), similar as in controls (D, E). (H, I) Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA, and cells were incubated at 37°C (H) or for 30 min at 38.5°C (I) before fixation. Here, green fluorescence surrounds the otoferlin immunofluorescence (arrows), suggesting loss of otoferlin from the plasma membrane.

J. t‐test; **P < 0.01). Quantification of the relative plasma membrane levels of otoferlin (as in Fig 1 ) revealed normal plasma membrane abundance when the human RXR motif was present, but a strong reduction for otoferlin with RXR and Ile515Thr (individual cells; mean ± SEM;‐test; **< 0.01).

A 20 amino acid stretch present in human otoferlin reduces plasma membrane localization of Ile515Thr‐otoferlin Looking at differences in the protein sequence of human and mouse otoferlin which might contribute to the less pronounced heat sensitivity in mice, we found an arginine‐rich insertion including an RXR motif at position 1244–1263 in the long human reference sequences (e.g. variant e; NP_001274418). While there is currently no experimental evidence which splice isoform regarding this motif is expressed in human IHCs, PCRs on mouse organ of Corti cDNA revealed that the isoform lacking this RXR motif is predominant in mouse IHCs (Fig EV4). A similar motif was described to cause a temperature‐dependent decrease in surface expression of an alpha‐adrenergic receptor (Filipeanu et al, 2011, 2015). We subcloned the human RXR stretch into mouse otoferlin cDNA and performed biolistic transfection into Otof−/− IHCs (Fig 5E–I). RXR‐otoferlin showed a strong plasma membrane immunostaining (relative to total cellular otoferlin levels), which was comparable to the values from explanted Otof+/+ cells (Fig 5D and J). Otoferlin with both the Ile515Thr mutation and the RXR motif, however, displayed almost complete absence from the plasma membrane already at 37°C (Fig 5H–J), which is in stark contrast to mouse Ile515Thr‐otoferlin (Fig 1). Since the abundance of otoferlin at the plasma membrane seems to be most relevant for sound encoding in vivo, this might explain the more pronounced heat sensitivity in human patients. When comparing to the hearing phenotype of our mouse models, hearing at normal and elevated temperature in human Ile515Thr patients would best be explained by a mixture of the splice variant with RXR and the splice variant without being expressed in human IHCs. Click here to expand this figure. Figure EV4.Sequence variations in mouse and human otoferlin and comparison with other species CLUSTAL Omega multiple sequence alignment for otoferlin homologues performed with the following sequences: Human otoferlin isoform e, NP_001274418.1; Bos Taurus, NP_001137579.1; Rattus norvegicus, NP_001263649; Mus musculus transcript variant 4, NM_001313767.1 (cDNA used in our experiments); Mus musculus transcript variant 1, NP_001093865.1; Mus musculus transcript variant 2, NP_114081.2; Xenopus tropicalis, XP_012826776.1. The presence of the 15 amino acid stretch SKG…GEH was tested in a PCR in (C). Sequence alignment, also including human otoferlin isoform b, NP_004793. Focus on the amino acid region between C 2 D and C 2 E bearing an arginine‐rich sequence in human otoferlin isoform e, including an RXR motif (in red). Labelled in yellow is the human sequence that was introduced by site‐directed mutagenesis into the mouse variant 4 for experiments in Fig 5. A PCR on mouse organ of Corti cDNA from P14 animals was performed to test for the expression of the mouse sequence variations. Left lane: using the primers AAGGACAGCCAGGAGACAGA and ATCTTGTCTTTGGGGCTCCT that bind before and after amino acid 168 in mouse variant 4 was used to distinguish between splice variants. For transcript variants 2 and 4, an amplicon of 155 bp was expected, whereas variant 1 should give rise to a 200‐bp amplicon. The PCR indicates that almost exclusively the short variant is transcribed. Right lane: using primers TCATCTACCGACCTCCAGACC and CACATCCACCTTGACCACAGC binding before and after amino acid 1242 in mouse variant 4 expected to lead to an amplicon size of 148 bp for variants 1 and 4 or 208 bp for variant 2. The strong band at 148 bp indicates that the vast majority of cDNA molecules confirmed the expression of the short variant. In conclusion, our data indicate that transcript variant 4 seems to be the predominantly transcribed otoferlin isoform in mouse organs of Corti.

Heat reduces exocytosis and otoferlin membrane levels We next tested for a synaptic dysfunction elicited by fever by performing patch clamp recordings of IHCs (Fig 6). We recorded ΔC m in IHCs induced by 5–100 ms depolarization steps, first at room temperature (RT), then at near physiological temperature (PT, 35–36.5°C), followed by high temperature (38.5–40°C). Consistent with a previous study (Nouvian, 2007), we found Ca2+ currents and exocytosis from Otof+/+ IHCs to increase when temperature was raised from RT to PT (Fig 6A and B). While Ca2+ current integrals increased only by 27 and 15% in Otof+/+ (n = 4) and OtofI515T/I515T (n = 5), respectively, exocytosis was more strongly enhanced by warming (Fig 6B) and increased 2.7‐ to threefold in both genotypes. Figure 6.Temperature alters exocytosis, otoferlin protein levels and plasma membrane abundance A–E. Capacitance increments recorded from Otof +/+ (black) and Otof I515T / I515T (red) IHCs. Individual cells recorded at indicated temperatures (light circles), mean ± SEM (filled circles) for perforated‐patch clamp experiments.

F, G. Summary of the capacitance changes and Ca 2+ current integrals for Otof +/+ (F) and Otof I515T / I515T IHCs (G) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature. Significant differences compared to RT measurements are indicated with colours of the respective temperature, and between PT and high temperature in violet.

H–J. Otoferlin immunofluorescence in Otof +/+ IHCs (upper panel) and Otof I515T / I515T IHCs (middle and lower panels) of explanted organs of Corti at P7‐P8 after incubation at indicated temperatures for 24 h; maximum projections of z‐stacks, inverted images; scale bar, 10 μm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.

K. Quantification of otoferlin immunofluorescence in Otof +/+ IHCs (black bars) and Otof I515T / I515T IHCs (red bars) indicates reduced protein levels with increasing temperature.

L. Apical/basal otoferlin protein distribution, revealing a significant apical shift of otoferlin and Vglut3 for Otof I515T / I515T IHCs (red symbols) at 38.5°C compared to Otof +/+ (grey/black symbols).

M. Relative levels of membrane‐bound otoferlin were lowered with increasing temperature.

N. Absolute otoferlin membrane immunostaining strongly decreased with temperature. Data information: All data presented are mean ± SEM. n = 98–137 Otof+/+cells and n = 90–97 OtofI515T/I515T cells in (K–N), 4–5 experiments. t‐test (A–G) or Kruskal–Wallis test (K–N); *P < 0.05; **P < 0.01; ***P < 0.001. Data information: All data presented are mean ± SEM.= 98–137cells and= 90–97cells in (K–N), 4–5 experiments.‐test (A–G) or Kruskal–Wallis test (K–N); *< 0.05; **< 0.01; ***< 0.001. We next heated the bath to 38.5–40°C expecting a strong reduction of exocytosis for OtofI515T/I515T IHCs given the human fever‐induced deafness. Recordings were started as soon as the temperature recorded in the bath close to the tissue was stable. Surprisingly, we found a strong decline in ΔC m for Otof+/+ IHCs compared to PT, especially for sustained exocytosis which was halved (Fig 6C and F). This fever‐induced reduction was weaker in OtofI515T/I515T (13% less than at PT; Fig 6G). As a result, ΔC m did not differ between OtofI515T/I515T and Otof+/+ IHCs at high temperature for all depolarization durations tested (Fig 6C). However, we cannot exclude that a longer heating period might have unravelled a stronger synaptic dysfunction in OtofI515T/I515T IHCs. In fact, sustained exocytosis between the genotypes appeared slightly stronger in recordings after heating. At 2 min of recovery at < 29°C, exocytosis in Otof+/+ IHCs was reduced to 52% relative to RT before heating for depolarization stimuli of ≥ 10 ms (n = 4), while in OtofI515T/I515T IHCs, it was reduced to 40% of initial RT values (n = 3; Fig 6D, F and G). Ca2+ currents were of similar size as before heating and comparable between Otof+/+ and OtofI515T/I515T IHCs. In few of these IHCs, exocytosis was tested also after a longer recovery period of 5–20 min, and we recorded additional cells only after heating (Fig 6E). Considering IHCs of both conditions, we found ΔC m levels for 100 ms depolarization in Otof+/+ IHCs to be restored to 61% compared to observations at RT before heating (n = 7 from 4 cells). In contrast, in OtofI515T/I515T IHCs, sustained exocytosis remained reduced (37% of initial values for 100 ms depolarization, n = 8 from 4 cells). Based on the loss of exocytic capacity in wild‐type and mutant IHCs between PT and ≥ 38.5°C, we propose that part of the synaptic machinery is thermally unstable. The poorer recovery from heat in OtofI515T/I515T IHCs and the temperature‐sensitive phenotype of patients with different OTOF mutations suggest that otoferlin itself is the heat‐sensitive protein and that heat instability of otoferlin is enhanced by certain mutations like Ile515Thr. Indeed, 3D structure predictions show that the isoleucine 515 points towards the hydrophobic core of the C 2 C domain (Appendix Fig S3), suggesting that an exchange into the more hydrophilic threonine potentially decreases the stability of the protein. Since we also did not find a change in otoferlin mRNA levels in OtofI515T/I515T organs of Corti, ruling out lower transcription levels or destabilized mRNA as a cause (Appendix Fig S4A), we hypothesized that mutations in otoferlin lead to faster protein degradation at elevated temperature. Testing the degradation rate of a series of otoferlin mutants related to human heat‐induced deafness after heterologous expression in HEK293T cells, we found hardly any degradation within 2 or 24 h even at elevated temperature, similar as for wild‐type otoferlin (Appendix Fig S4B and C). Although the lifetime of mutated otoferlin might be shorter in IHCs (see Appendix Fig S1E), the degradation seems too slow to explain hearing loss due to elevation of the body temperature. We hypothesize that loss of otoferlin from the plasma membrane in addition to protein unfolding might explain acute IHC presynaptic dysfunction at fever. Next, we tested steady‐state levels and subcellular distribution of otoferlin after prolonged incubation at elevated temperature (Fig 6H–J). In cultures of P7‐8 organs of Corti incubated for 24 h at 37°C, the physiological temperature of mice (http://www.informatics.jax.org), otoferlin protein levels in OtofI515T/I515T IHCs were reduced to 29% of Otof+/+ controls at 37°C (Fig 6K). After 24‐h incubation at 38.5°C, otoferlin protein levels were even further reduced to 21%, and, remarkably, both otoferlin and Vglut3 localized more apically in OtofI515T/I515T IHCs (Fig 6L), resembling the distribution found in OtofPga/Pga IHCs (Fig 1H). The relative levels of plasma membrane‐bound otoferlin were strongly reduced at febrile temperature (56% of controls, Fig 6M), resulting in a strong temperature‐dependent reduction of the absolute amount of plasma membrane‐bound otoferlin in OtofI515T/I515T IHCs (12% of controls, Fig 6N). In summary, otoferlin is sensitive to heat, which is exacerbated by mutations like Ile515Thr. We found indications for faster degradation and loss from the plasma membrane of Ile515Thr‐otoferlin at febrile temperature.