RNA base editing therapy cures hearing loss induced by OTOF gene mutation.

Otoferlin (OTOF) gene mutations represent the primary cause of hearing impairment and deafness in auditory neuropathy. The c.2485C>T (p. Q829X) mutation variant is responsible for approximately 3% of recessive prelingual deafness cases within the Spanish population. Previous studies have used two recombinant AAV vectors to overexpress OTOF, albeit with limited efficacy. In this study, we introduce an enhanced mini-dCas13X RNA base editor (emxABE) delivered via an AAV9 variant, achieving nearly 100% transfection efficiency in inner hair cells. This approach is aimed at treating OTOFQ829X, resulting in an approximately 80% adenosine-to-inosine conversion efficiency in humanized OtofQ829X/Q829X mice. Following a single scala media injection of emxABE targeting OTOFQ829X (emxABE-T) administered during the postnatal day 0-3 period in OtofQ829X/Q829X mice, we observed OTOF expression restoration in nearly 100% of inner hair cells. Moreover, auditory function was significantly improved, reaching similar levels as in wild-type mice. This enhancement persisted for at least 7 months. We also investigated P5-P7 and P30 OtofQ829X/Q829X mice, achieving auditory function restoration through round window injection of emxABE-T. These findings not only highlight an effective therapeutic strategy for potentially addressing OTOFQ829X-induced hearing loss but also underscore emxABE as a versatile toolkit for treating other monogenic diseases characterized by premature termination codons.

Impaired Hearing and Altered Subplate Circuits During the First and Second Postnatal Weeks of Otoferlin-Deficient Mice.

Sensory deprivation from the periphery impacts cortical development. Otoferlin deficiency leads to impaired cochlear synaptic transmission and is associated with progressive hearing loss in adults. However, it remains elusive how sensory deprivation due to otoferlin deficiency impacts the early development of the auditory cortex (ACX) especially before the onset of low threshold hearing. To test that, we performed in vivo imaging of the ACX in awake mice lacking otoferlin (Otof-/-) during the first and second postnatal weeks and found that spontaneous and sound-driven cortical activity were progressively impaired. We then characterized the effects on developing auditory cortical circuits by performing in vitro recordings from subplate neurons (SPN), the first primary targets of thalamocortical inputs. We found that in Otof-/- pups, SPNs received exuberant connections from excitatory and inhibitory neurons. Moreover, as a population, SPNs showed higher similarity with respect to their circuit topology in the absence of otoferlin. Together, our results show that otoferlin deficiency results in impaired hearing and has a powerful influence on cortical connections and spontaneous activity in early development even before complete deafness. Therefore, peripheral activity has the potential to sculpt cortical structures from the earliest ages, even before hearing impairment is diagnosed.

Increased Otoferlin Expression in B Cells Is Associated with Muscle Weakness in Untreated Juvenile Dermatomyositis: A Pilot Study.

Otoferlin mRNA expression is increased in JDM patients' PBMCs and muscle compared to healthy controls. This study aims to evaluate the role of otoferlin in JDM disease pathophysiology and its association with disease activity in untreated children with JDM. A total of 26 untreated JDM (88.5% female, 92.3% white, non-Hispanic) and 15 healthy controls were included in this study. Otoferlin mRNA expression was determined by qRT-PCR before and a few months after therapy. Detailed flow cytometry of various cell surface markers and cytoplasmic otoferlin was performed to identify cells expressing otoferlin. In addition, muscle otoferlin expression was evaluated in situ in six untreated JDM patients and three healthy controls. There was a significant increase in otoferlin expression in JDM children compared to controls (Median 67.5 vs. 2.1; p = 0.001). There was a positive correlation between mRNA otoferlin expression and the following disease activity markers: disease activity scores (DAS)-total (rs = 0.62, p < 0.001); childhood myositis assessment scale (CMAS) (rs = -0.61, p = 0.002); neopterin (rs = 0.57, p = 0.004) and von Willebrand factor antigen (vWF: Ag) (rs = 0.60, p = 0.004). Most of the otoferlin-positive cells were unswitched B cells (63-99.4%), with 65-75% of them expressing plasmablast markers (CD19+, IgM+, CD38hi, CD24-). The findings of this pilot study suggest that otoferlin expression is associated with muscle weakness, making it a possible biomarker of disease activity. Additionally, B cells and plasmablasts were the primary cells expressing otoferlin.

Predicting the Impact of OTOF Gene Missense Variants on Auditory Neuropathy Spectrum Disorder.

Auditory neuropathy spectrum disorder (ANSD) associated with mutations of the OTOF gene is one of the common types of sensorineural hearing loss of a hereditary nature. Due to its high genetic heterogeneity, ANSD is considered one of the most difficult hearing disorders to diagnose. The dataset from 270 known annotated single amino acid substitutions (SAV) related to ANSD was created. It was used to estimate the accuracy of pathogenicity prediction using the known (from dbNSFP4.4) method and a new one. The new method (ConStruct) for the creation of the protein-centric classification model is based on the use of Random Forest for the analysis of missense variants in exons of the OTOF gene. A system of predictor variables was developed based on the modern understanding of the structure and function of the otoferlin protein and reflecting the location of changes in the tertiary structure of the protein due to mutations in the OTOF gene. The conservation values of nucleotide substitutions in genomes of 100 vertebrates and 30 primates were also used as variables. The average prediction of balanced accuracy and the AUC value calculated by the 5-fold cross-validation procedure were 0.866 and 0.903, respectively. The model shows good results for interpreting data from the targeted sequencing of the OTOF gene and can be implemented as an auxiliary tool for the diagnosis of ANSD in the early stages of ontogenesis. The created model, together with the results of the pathogenicity prediction of SAVs via other known accurate methods, were used for the evaluation of a manually created set of 1302 VUS related to ANSD. Based on the analysis of predicted results, 16 SAVs were selected as the new most probable pathogenic variants.

Temperature-Sensitive Auditory Neuropathy: Report of a Novel Variant of OTOF Gene and Review of Current Literature.

Background and objectives: Otoferlin is a multi-C2 domain protein implicated in neurotransmitter-containing vesicle release and replenishment of the cochlear inner hair cell (IHC) synapses. Mutations in the OTOF gene have been associated with two different clinical phenotypes: a prelingual severe-to-profound sensorineural hearing loss (ANSD-DFNB9); and the peculiar temperature-sensitive auditory neuropathy (TS-ANSD), characterized by a baseline mild-to-moderate hearing threshold that worsens to severe-to-profound when the body temperature rises that returns to a baseline a few hours after the temperature has fallen again. The latter clinical phenotype has been described only with a few OTOF variants with an autosomal recessive biallelic pattern of inheritance. Case report: A 7-year-old boy presented a picture compatible with TS-ANSD exacerbated by febrile states or physical exercise with mild-to-moderate hearing loss at low and medium frequencies and a decrease in speech discrimination that worsened with an unfavorable speech-to-noise ratio. Otoacoustic emissions (OAEs) were present whereas auditory brainstem responses (ABRs) evoked by a click or tone-burst were generally absent. No inner ear malformations were described from the CT scan or MRI. Next-generation sequencing (NGS) of the known deafness genes and multi-phasic bioinformatic analyses of the data detected in OTOF a c.2521G>A missense variant and the deletion of 7.4 Kb, which was confirmed by array-comparative genomic hybridization (array-CGH). The proband's parents, who were asymptomatic, were tested by Sanger sequencing and the father presented the c.2521G>A missense variant. Conclusions: The picture presented by the patient was compatible with OTOF-induced TS-ANSD. OTOF has been generally associated with an autosomal recessive biallelic pattern of inheritance; in this clinical report, two pathogenic variants never previously associated with TS-ANSD were described.

Association of R1939W and P1987R variants of Otoferlin (OTOF) gene with severe to profound nonsyndromic sensorineural hearing loss in Pakistani subjects.

Objective: To find possible association of R1939W and P1987R variants of OTOF gene with severe to profound NSSHL in cochlear implant subjects. Methods: It was a case control study, conducted from June 2021 to February 2022, comprising 50 cases of severe to profound NSSHL who had received cochlear implant from ENT Department, CMH Rawalpindi and 50 age-matched healthy controls from PEMH Rawalpindi. Blood samples were collected from all the subjects, followed by DNA extraction and allele-specific polymerase chain reaction, performed at Multi-disciplinary Laboratory of Department of Biochemistry and Molecular Biology, Army Medical College Rawalpindi. Statistical analysis was done using 'SPSS' and 'XLSTAT', followed by genetic analysis using 'SNPstat'. Results: Mean age of the cases was 5.96 ± 4.62 years (N=50), comprising 58% males and 42% females. All had bilateral and prelingual HL. Parental consanguinity was 72%, whereas 62% cases had a positive family history of deafness. Alleles of R1939W and P1987R were not associated with NSSHL, as shown by their p values of 0.56 and 0.89 respectively. For R1939W ORs were 0.71 (dominant model) and 0.80 (overdominant model), indicating negative association with NSSHL. Regarding P1987R OR was 0.96 (log-additive model). Genotypes of both variants were not in HW Equilibrium (p <0.0001), whereas their alleles showed high LD (D'=0.92). Conclusion: High percentage of parental consanguinity was observed among cochlear implant candidates. The OTOF variants R1939W and P1987R were found to have protective roles against NSSHL in study population.

Development of synaptic fidelity and action potential robustness at an inhibitory sound localization circuit: effects of otoferlin-related deafness.

Sound localization involves information analysis in the lateral superior olive (LSO), a conspicuous nucleus in the mammalian auditory brainstem. LSO neurons weigh interaural level differences (ILDs) through precise integration of glutamatergic excitation from the cochlear nucleus (CN) and glycinergic inhibition from the medial nucleus of the trapezoid body (MNTB). Sound sources can be localized even during sustained perception, an accomplishment that requires robust neurotransmission. Virtually nothing is known about the sustained performance and the temporal precision of MNTB-LSO inputs after postnatal day (P)12 (time of hearing onset) and whether acoustic experience guides development. Here we performed whole-cell patch-clamp recordings to investigate neurotransmission of single MNTB-LSO fibres upon sustained electrical stimulation (1-200 Hz/60 s) at P11 and P38 in wild-type (WT) and deaf otoferlin (Otof) knock-out (KO) mice. At P11, WT and KO inputs performed remarkably similarly. In WTs, the performance increased drastically between P11 and P38, e.g. manifested by an 8 to 11-fold higher replenishment rate (RR) of synaptic vesicles and action potential robustness. Together, these changes resulted in reliable and highly precise neurotransmission at frequencies ≤100 Hz. In contrast, KO inputs performed similarly at both ages, implying impaired synaptic maturation. Computational modelling confirmed the empirical observations and established a reduced RR per release site for P38 KOs. In conclusion, acoustic experience appears to contribute massively to the development of reliable neurotransmission, thereby forming the basis for effective ILD detection. Collectively, our results provide novel insights into experience-dependent maturation of inhibitory neurotransmission and auditory circuits at the synaptic level. KEY POINTS: Inhibitory glycinergic inputs from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) are involved in sound localization. This brainstem circuit performs reliably throughout life. How such reliability develops is unknown. Here we investigated the role of acoustic experience on the functional maturation of MNTB-LSO inputs at juvenile (postnatal day P11) and young adult ages (P38) employing deaf mice lacking otoferlin (KO). We analysed neurotransmission at single MNTB-LSO fibres in acute brainstem slices employing prolonged high-frequency stimulation (1-200 Hz/60 s). At P11, KO inputs still performed normally, as manifested by normal synaptic attenuation, fidelity, replenishment rate, temporal precision and action potential robustness. Between P11 and P38, several synaptic parameters increased substantially in wild-type mice, collectively resulting in high-fidelity and temporally precise neurotransmission. In contrast, maturation of synaptic fidelity was largely absent in KOs after P11. Collectively, reliable neurotransmission at inhibitory MNTB-LSO inputs develops under the guidance of acoustic experience.

Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model.

Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5' and the other the 3' portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea of Otof-/- mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5' and 3' cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.

Otoferlin is a prognostic biomarker in patients with clear cell renal cell carcinoma: A systematic expression analysis.

OBJECTIVES: To comprehensively investigate the role of otoferlin as a prognostic and diagnostic biomarker in clear cell renal cell carcinoma. METHODS: Three independent cohorts were used to study otoferlin in clear cell renal cell carcinoma: The Cancer Genome Atlas cohort (messenger ribonucleic acid expression; clear cell renal cell carcinoma n = 514, normal renal tissue n = 81); study validation cohort (messenger ribonucleic acid expression; clear cell renal cell carcinoma n = 79, normal renal tissue n = 44); and immunohistochemistry cohort (protein expression; clear cell renal cell carcinoma n = 142, normal renal tissue n = 30). Otoferlin gene expressions were extracted from The Cancer Genome Atlas database or determined using quantitative real-time polymerase chain reaction, respectively. Protein expression was assessed using immunohistochemistry staining against otoferlin on tissue microarrays. Correlations between otoferlin messenger ribonucleic acid/protein expression and clinicopathological data/patient survival were statistically tested. RESULTS: Otoferlin messenger ribonucleic acid expression was significantly upregulated in clear cell renal cell carcinoma compared with normal renal tissue. High expression levels correlated with advanced stage, higher grade and metastatic tumors, accompanied by independent prognostic significance for overall and cancer-specific survival. In contrast, otoferlin protein expression was downregulated in tumor tissue. Although, high otoferlin expression in clear cell renal cell carcinoma was positively correlated with histological grading and independently predictive of a shortened progression-free survival. CONCLUSION: Our data suggest otoferlin as an indicator of tumor aggressiveness and as a prognostic biomarker for patients with clear cell renal cell carcinoma, leading to the conclusion that otoferlin could promote the malignancy of clear cell renal cell carcinoma.

Viral Transfer of Mini-Otoferlins Partially Restores the Fast Component of Exocytosis and Uncovers Ultrafast Endocytosis in Auditory Hair Cells of Otoferlin Knock-Out Mice.

Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Cav1.3 calcium channels. At these synapses, the fast and indefatigable release of synaptic vesicles by IHCs is controlled by otoferlin, a six-C2-domain (C2-ABCDEF) protein that functions as a high-affinity Ca2+ sensor. The molecular events by which each otoferlin C2 domain contributes to the regulation of the synaptic vesicle cycle in IHCs are still incompletely understood. Here, we investigate their role using a cochlear viral cDNA transfer approach in vivo, where IHCs of mouse lacking otoferlin (Otof-/- mice of both sexes) were virally transduced with cDNAs of various mini-otoferlins. Using patch-clamp recordings and membrane capacitance measurements, we show that the viral transfer of mini-otoferlin containing C2-ACEF, C2-EF, or C2-DEF partially restores the fast exocytotic component in Otof-/- mouse IHCs. The restoration was much less efficient with C2-ACDF, underlining the importance of the C2-EF domain. None of the mini-otoferlins tested restored the sustained component of vesicle release, explaining the absence of hearing recovery. The restoration of the fast exocytotic component in the transduced Otof-/- IHCs was also associated with a recovery of Ca2+ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Cav1.3 channels. Finally, the reintroduction of the mini-otoferlins C2-EF, C2-DEF, or C2-ACEF allowed us to uncover and characterize for the first time a dynamin-dependent ultrafast endocytosis in IHCs.SIGNIFICANCE STATEMENT Otoferlin, a large six-C2-domain protein, is essential for synaptic vesicle exocytosis at auditory hair cell ribbon synapses. Here, we show that the viral expression of truncated forms of otoferlin (C2-EF, C2-DEF, and C2-ACEF) can partially rescue the fast and transient release component of exocytosis in mouse hair cells lacking otoferlin, yet cannot sustain exocytosis after long repeated stimulation. Remarkably, these hair cells also display a dynamin-dependent ultrafast endocytosis. Overall, our study uncovers the pleiotropic role of otoferlin in the hair cell synaptic vesicle cycle, notably in triggering both ultrafast exocytosis and endocytosis and recruiting synaptic vesicles to the active zone.

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

The multi-C2 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, OtofI515T/I515T, exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In OtofI515T/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.

Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels.

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins.

Effects of imidacloprid, a neonicotinoid insecticide, on the echolocation system of insectivorous bats.

Imidacloprid, a widely used neonicotinoid insecticide, has led to a decline in the honey bee population worldwide. An invertebrate insect prey with neonicotinoid toxicity can adversely affect insectivores, such as echolocating bats. The aim of the current study was to examined whether imidacloprid toxicity may interfere echolocation system such as vocal, auditory, orientation, and spatial memory systems in the insectivorous bat. By comparing the ultrasound spectrum, auditory brainstem-evoked potential, and flight trajectory, we found that imidacloprid toxicity may interfere functions in vocal, auditory, orientation, and spatial memory system of insectivorous bats (Hipposideros armiger terasensis). As suggested from immunohistochemistry and western blots evidences, we found that insectivorous bats after suffering imidacloprid toxicity may decrease vocal-related FOXP2 expressions in the superior colliculus, auditory-related prestin expressions in the cochlea, and the auditory-related otoferlin expressions in the cochlea and the inferior colliculus, and cause inflammation and mitochondrial dysfunction-related apoptosis in the hippocampal CA1 and medial entorhinal cortex. These results may provide a reasonable explanation about imidacloprid-induced interference of echolocation system in insectivorous bats.

Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness.

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In CaV1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

[OTOF-related auditory neuropathy spectrum disorder]. / Zabolevanie spektra auditornykh neiropatii, obuslovlennoe mutatsiyami v gene otoferlina (OTOF).

Otoferlin (OTOF) gene mutations are the most common cause of hereditary ANSD according to investigations in several countries. THE AIM: Of this study was to estimate the prevalence of OTOF mutations in Russian children with ANSD and evaluate audiological and clinical features of OTOF-related ANSD. PATIENTS AND METHODS: 28 children with bilateral ANSDwere enrolled in the investigation. Two step genetic testing was performed: first step - GJB2 gene testing to exclude GJB2-related hearing loss; second step - NGS-based sequencing to explore another 35 hearing loss genes (including OTOF). RESULTS: OTOF mutations, including 6 new variants, were found in 5 children with ANSD (18%). All 5 children had no risk factors for hearing loss and passed hearing screening. OAE and cochlear microphonics were present till the last testing at the age of 4-5 years. ABR were not detectable. The ASSR were measurable bilaterally at all frequencies in all cases, but they did not correlate with behavioral thresholds that revealed severe hearing loss. Hearing thresholds were stable during follow up period. 3 children underwent cochlear implantation. After cochlear implantation auditory nerve action potentials to electric stimulation were detected within normal range. CONCLUSION: Genetic testing of children with ANSD and first of all OTOF testing enables to reveal hearing loss etiology and provide the optimal rehabilitation approach, including cochlear implantation, as early as possible.

Topographic map refinement and synaptic strengthening of a sound localization circuit require spontaneous peripheral activity.

KEY POINTS: Loss of the calcium sensor otoferlin disrupts neurotransmission from inner hair cells. Central auditory nuclei are functionally denervated in otoferlin knockout mice (Otof KOs) via gene ablation confined to the periphery. We employed juvenile and young adult Otof KO mice (postnatal days (P)10-12 and P27-49) as a model for lacking spontaneous activity and deafness, respectively. We studied the impact of peripheral activity on synaptic refinement in the sound localization circuit from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO). MNTB in vivo recordings demonstrated drastically reduced spontaneous spiking and deafness in Otof KOs. Juvenile KOs showed impaired synapse elimination and strengthening, manifested by broader MNTB-LSO inputs, imprecise MNTB-LSO topography and weaker MNTB-LSO fibres. The impairments persisted into young adulthood. Further functional refinement after hearing onset was undetected in young adult wild-types. Collectively, activity deprivation confined to peripheral protein loss impairs functional MNTB-LSO refinement during a critical prehearing period. ABSTRACT: Circuit refinement is critical for the developing sound localization pathways in the auditory brainstem. In prehearing mice (hearing onset around postnatal day (P)12), spontaneous activity propagates from the periphery to central auditory nuclei. At the glycinergic projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) of neonatal mice, super-numerous MNTB fibres innervate a given LSO neuron. Between P4 and P9, MNTB fibres are functionally eliminated, whereas the remaining fibres are strengthened. Little is known about MNTB-LSO circuit refinement after P20. Moreover, MNTB-LSO refinement upon activity deprivation confined to the periphery is largely unexplored. This leaves a considerable knowledge gap, as deprivation often occurs in patients with congenital deafness, e.g. upon mutations in the otoferlin gene (OTOF). Here, we analysed juvenile (P10-12) and young adult (P27-49) otoferlin knockout (Otof KO) mice with respect to MNTB-LSO refinement. MNTB in vivo recordings revealed drastically reduced spontaneous activity and deafness in knockouts (KOs), confirming deprivation. As RNA sequencing revealed Otof absence in the MNTB and LSO of wild-types, Otof loss in KOs is specific to the periphery. Functional denervation impaired MNTB-LSO synapse elimination and strengthening, which was assessed by glutamate uncaging and electrical stimulation. Impaired elimination led to imprecise MNTB-LSO topography. Impaired strengthening was associated with lower quantal content per MNTB fibre. In young adult KOs, the MNTB-LSO circuit remained unrefined. Further functional refinement after P12 appeared absent in wild-types. Collectively, we provide novel insights into functional MNTB-LSO circuit maturation governed by a cochlea-specific protein. The central malfunctions in Otof KOs may have implications for patients with sensorineuronal hearing loss.

Ferlins Show Tissue-Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans-Golgi/Recycling Ferlins.

Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+)-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+)-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+)-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca2+] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca2+ Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by KD values from SPR (+Ca2+), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted.

Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.